Fig. 5

Fig. 5

(A) Wnt5a blocked the degradation of endogenous Vangl proteins. HEK293T cells were treated with CHX (100 μg/ml) alone or with rWnt5a (200 ng/ml) for the indicated time. The relative Vangl protein levels are quantified on the bottom panel. (B) Wnt5a inhibited KBTBD7-induced Vangl2 ubiquitination through CK1-mediated Vangl2 phosphorylation. CHO cells were treated with or without CK1 inhibitor D4476 (100 μM) for 6 hours. (C) Vangl2 phosphorylation inhibited its ubiquitination. HEK293T cells expressing WT, phospho-mutant (A), or phospho-mimetic (E) Vangl2 were treated with MG132 (10 μM) for 4 hours. Because phospho-mutant Vangl2 was known to be unstable, more plasmids were transfected to increase the input level. (D) CK1 protected Vangl2 from the KBTBD7-induced degradation. (E) Wnt5a and CK1 induced Vangl2 basal phosphorylation in the ER. CHO cells were treated with rWnt5a (200 ng/ml) for 2 hours or transfected with CK1δ. Vangl2 was examined in total cell lysates (T) and ER fractions (ER). ATF6 served as an ER marker. The lower black, middle blue, and upper red arrows point to the unphosphorylated, basal phosphorylated, and hyperphosphorylated Vangl2, respectively. The percentage of basal phosphorylated Vangl2 (blue) and unphosphorylated Vangl2 (black) in the ER fractions is quantified in the bottom panel. (F) Phosphorylation facilitated ER export of Vangl2. The transient expression of GFP-HA-tagged WT, phospho-mutant (A), phospho-mimetic (E), VIM mutant (R334A), or Lp mutant (S464N) Vangl2 (green) was induced by doxycycline (1 μg/ml) in MDCK stable cell lines for 1 or 2 hours (upper two rows). Doxycycline was removed after 2 hours of treatment, and the fate of Vangl2 (green) was traced for another 2 or 4 hours (lower two rows). ER, ER dye (red); nuclei, DAPI (blue). Scale bar, 10 μm.